Polyglycerol and lactose compositions for the protection of living systems from states of reduced metabolism

ABSTRACT

Polyglycerol, lactose, and a combination of polyglycerol and lactose are effective at preserving cells, tissues, and organs from damage due to hypothermic, ischemic, or other metabolic impairment, and a mixture of polyglycerol plus lactose is particularly useful for the hypothermic storage of cells, tissues, and organs. The mixture of polyglycerol and lactose can be further improved by the addition of chondroitin sulfate, chlorpromazine, calcium, citrate, glutathione, adenine, glucose, magnesium, and a pH buffer.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.10/066,285, filed Feb. 1, 2002 which is a Continuation in Part of U.S.patent application Ser. No. 09/726,857, filed Nov. 30, 2000 which claimspriority under 35 U.S.C. § 119 of U.S. Provisional Application No.60/167,963, filed Nov. 30, 1999 (herein incorporated by reference). Thisapplication is also a Continuation in Part of U.S. patent applicationSer. No. 09/916,396, filed Jul. 27, 2001, which claims priority under 35U.S.C. § 119 of U.S. Provisional application 60/221,691, filed Jul. 31,2000, all of which are herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates generally to the field of cell, tissue, and organpreservation. More specifically, this invention relates to the field ofprotection of cells, tissues, and organs from states such ashypothermia. Still more specifically, this invention relates to the useof polyglycerol and other solutes, especially alpha lactose, forprotecting living systems during periods of depressed metabolism, withor without supercooling.

BACKGROUND OF THE INVENTION

The current shortage of organs for transplantation mandates that maximumusage be obtained from the scarce resource represented by vital organs.Despite this imperative, many organs that could be transplanted are nottransplanted due to limitations on the useful lifetime of organs afterthey are removed from the body. Thus, there is clearly a need for betterpreservation solutions for vital organs.

Many preservation solutions have been proposed and patented in the priorart. However, the primary solution used worldwide for mosttransplantable organs is UW Solution, also known as VIASPAN™, the tradename under which it was initially sold by DuPont Pharmaceuticals. Thereis no alternative solution that is generally regarded as being superiorto UW Solution. Therefore, to be useful, any new organ preservationsolutions should be at least competitive with UW Solution.

Despite its widespread use, there is much dissatisfaction with UWSolution as an organ preservation solution. First of all, it requiresthe addition of expensive additives prior to use, which is inconvenient.Second, UW has to be washed out prior to transplantation of thepreserved organ, which is also inconvenient and wastes valuableoperating room time. Third, UW solution is viscous and sticky andtherefore does a poor job of washing blood out of organs that areflushed with it. To respond to this problem, many centers flush out theblood with a different solution and then flush out this intermediatesolution with UW, again wasting time and solution. Alternatively, bloodremaining in the organ may account for the failure of a large fractionof transplanted kidneys to function immediately upon transplantation.Fourth, kidneys preserved with UW Solution by simple cold storage forover 24 hours are sufficiently damaged to offset the advantages of organsharing for improving tissue matching between donor and recipient.Tissue matching is known to have major effects on survival rates five toten years following transplantation. Therefore, improved preservationafter 24 hours of storage could translate into expanded use ofwell-matched organ transplants and improved long-term survival of organrecipients. This in turn would mean the need for fewer re-transplantsafter rejection, which would free up the otherwise-needed organs forother recipients.

The limitations and poor efficacy of UW and other organ preservationsolutions for preservation for 24 hours and beyond indicate the need foran improved solution.

SUMMARY OF THE INVENTION

The present invention is a preservation solution for cells, tissues, andorgans comprising a combination of polyglycerol and lactose in an amounteffective to preserve said cells, tissues, and organs under hypothermicand other reduced-metabolism conditions. In one embodiment the lactoseis alpha lactose. In a further embodiment, the polyglycerol is from n=2to n=200 monomers. In a further embodiment, the polyglycerol isdecaglycerol or hexaglycerol. The lactose is preferably at aconcentration from about 11 mM to 250 mM, or more preferably at about20-150 mM. The polyglycerol is preferably at a concentration of 10 mOsmto 250 mOsm (about 0.3% w/v to about 7.3% w/v, respectfully, fordecaglycerol), or more preferably from about 20-200 mOsm. In a furtherembodiment, the preservation solution may additionally containchondroitin sulfate, preferably at a concentration of about 0.01% w/v to1% w/v. In a further embodiment, the preservation solution mayadditionally contain chlorpromazine, preferably at a concentration ofabout 1-50 micrograms/ml, even more preferably at about 2-10micrograms/ml. The sum of lactose+polyglycerol osmolality is preferablyfrom 20-250 mOsm.

In an alternative embodiment, the preservation solution may also containone or more of: calcium, citrate, glutathione, NaCl, at least onebuffer, such as a phosphate buffer, glucose, adenine, magnesium, andacetate.

In one embodiment, the solution has an osmolality of less than about 350mOsm. In a further embodiment, the sum of all impermeant speciescontributes 20-250 mOsm (milliosmolal) to the osmolality of thesolution.

A further embodiment is a method for the preservation of cells, tissues,or organs under conditions of impaired cell volume homeostasis inaddition to hypothermia below 10° C., which method involves contactingthe cells, tissues, or organs with a solution comprising polyglycerol inan amount effective to preclude or to reverse cell swelling. Thecontacting may be via intravenous or intra-arterial administration. Thecontacting may be in vivo via arterial organ perfusion or retrogradevenous perfusion of an organ or vascularized tissue. Alternatively, thecontacting may be in vitro via arterial organ perfusion or retrogradevenous perfusion of an organ or vascularized tissue. In one embodiment,the contacting is via the immersion of or bathing of affected cells,tissues, or organs. The polyglycerol is preferably from n=2 to 200monomer units in length and may be tetraglycerol, hexaglycerol, ordecaglycerol. Preferably the polyglycerol is at a concentration of fromabout 20 mOsm to 1,500 mOsm when in contact with said cell, tissue, ororgan. The preservation solution may also comprise lactose. In oneembodiment, the lactose is alpha lactose.

In a further embodiment, the effective amount is an isotonic solution.Alternatively, the effective amount is a hypertonic solution.

One embodiment is a method for preserving cells, tissues, or organsunder conditions of impaired cell volume homeostasis by contacting thecells with a solution comprising polyglycerol in an amount effective topreclude or to reverse cell swelling.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects and advantages of the presentinvention will now be described with reference to the drawings of thepreferred embodiment, which embodiment is intended to illustrate and notto limit the invention, and in which:

FIG. 1 shows the lack of toxicity of PGL as used in an organpreservation solution in place of glucose for preservation of kidneyslices.

FIG. 2 shows the lack of toxicity of PGL used in a perfusate employedfor the preservation of whole kidneys.

FIG. 3 shows postoperative serum creatinine levels in rabbits receivingkidneys preserved for 24 hrs at 0° C. with either solutions of thepresent invention or UW Solution (Viaspan).

FIG. 4 shows postoperative serum creatinine levels in rabbits receivingkidneys preserved for 24 hrs at 0° C. with solutions comprisingpolyglycerol without lactose, lactose without polyglycerol, or acombination of polyglycerol and lactose.

FIG. 5 shows postoperative serum creatinine levels in rabbits receivingkidneys preserved for 48 hrs at 0° C. with either solutions of thepresent invention or UW Solution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Polyglycerol (PGL) is a water-soluble, non-toxic, FDA-approved, lowmolecular weight polymer given by the formulaH[—OCH₂CHOHCH₂—]_(n)OH.

PGL is commercially available with n=2 (diglycerol) up to n=10(decaglycerol) and beyond. For tetraglycerol, n=4, and for hexaglycerol,n=6. The molecular mass of decaglycerol is about 758 daltons.

PGL is an inexpensive, non-toxic compound used in cosmetics and inesterified form as a food additive that can replace more than 50% of fatcalories in some food (Babayan, J. Environ. Pathol. Toxicol. Oncol.6:15-24, 1986). Inside the body, PGL esters are metabolized back intoPGL, which underscores the non-toxic nature of this compound. Consumersof MACDONALD'S donuts and birthday cakes, WEIGHT WATCHER'S ice cream,dietetic desserts and chocolate emulsified with polyglycerolpolyricinoleic acid, for example, have PGL released into their bodieswithout negative consequences.

Although this molecule was heretofore used only in the food industry asa sweetener and fat substitute, it was surprisingly found by one of thepresent inventors to possess the ability to prevent nucleation of ice insupercooled solutions (U.S. patent application Ser. No. 09/726,857).Given the potential usefulness of supercooling for the preservation ofcells, tissues, organs, and proteins near 0° C., (see, for example,Conn. Med. 59, pp. 387-99, 1995—herein incorporated in its entirety byreference thereto), it became of interest to know whether PGL could alsosupport the viability of cells, tissues, and organs at temperatures inthe vicinity of 0° C.

One of the cornerstones of organ preservation solutions is non-toxicimpermeants. The impermeants of UW Solution are raffinose, hydroxyethylstarch, and either lactobionate or gluconate. Although many otherimpermeants have been proposed and tested, there is no generallyaccepted impermeant known that is superior to the impermeants of UWSolution. It is an object of the present invention to provide solutionsfor the superior protection of living cells, tissues, and organs, saidsolutions having polyglycerol and/or lactose, in which, when combined,the proportion of polyglycerol to lactose may vary from 1:0 to 0:1, butin which the ratio of the osmolality of polyglycerol to that of lactoseis most preferably in the range of 0.2 to 10.

Lactose is a disaccharide composed of galactose and glucose. In nature,it is found only in milk. Because milk is produced in industrialquantities in the United States, rather pure alpha-lactose can bepurchased for as little as $9.90 per kilogram (Sigma Chemical 1999catalogue number L 3625), making it even less costly than sucrose. Otherforms of lactose (beta lactose, D-lactose, and allolactose) are alsoenvisioned to be effective, but are more costly than alpha lactose.

Lactose has never been explored as a component of an organ preservationsolution, and no information on its suitability was available until thepresent disclosure. Lactose is a reducing sugar, which previously mighthave made it seem undesirable. Further, UW Solution contains raffinose,a trisaccharide costing over $2000/kg, and has been sold commerciallyworldwide for over 15 years despite the price differential betweenraffinose and lactose. Clearly, this indicates that using lactose inplace of a higher molecular mass sugar such as raffinose or in place ofthe charged impermeants such as lactobionate or gluconate was notpreviously contemplated. This is logical based on a) the rational beliefin the prior art that a disaccharide will be inferior to a trisaccharidein its ability to preclude hypothermic cell swelling, and b) theobservation of many investigators that raffinose does not compete withlactobionate in importance in UW Solution, the raffinose beingdispensable without harm, in contrast to the indispensability oflactobionate. Further, as is described in FIG. 1 below, sucrose, themost popular disaccharide, was less effective than the monosaccharide,glucose, in supporting the viability of rabbit renal cortical slicesduring prolonged cold storage, despite the fact that it is anon-reducing disaccharide as opposed to glucose, which is a reducingmonosaccharide. The latter observation demonstrates that the efficacy ofsugars for cold storage solutions cannot be predicted, and must bedetermined experimentally. The choice of lactose has never beencontemplated in the prior art for use in cell preservation orprotection.

Polyglycerol and alpha lactose are non-toxic molecules that are unableto cross the cell membrane under hypothermic conditions and thereforeare capable of arresting cell swelling during cell, tissue, or organstorage. Furthermore, polyglycerol is expected to be impermeable even atelevated temperatures, such as normal body temperature (37° C.), andtherefore will protect cells, tissues, organs, and organisms from hightemperature states of reduced metabolism or reduced cell volumeregulatory ability, such as in ouabain poisoning, warm ischemia, shock,heat stroke, or cardiac arrest. Alpha lactose, polyglycerol, and thecombination thereof are also useful for protecting organs duringcooling, warming, and holding (for example, during transplantation) andmay also be efficacious for preservation by perfusion or intermittentperfusion as opposed to simple static storage.

It is not predictable that the combination of a disaccharide with thelarger decaglycerol molecule would improve the efficacy of decaglycerolfor inhibition of cell damage during cold storage, since decaglycerolalone should be fully sufficient for this role, and lactose should haveno added benefit. Nevertheless, surprisingly, the combination of alphalactose and decaglycerol is more efficacious than the use ofdecaglycerol alone when essentially equi-osmotic concentrations ofdecaglycerol or decaglycerol plus lactose are compared. By supplyingpolyglycerol in combination with lactose, with or without otherappropriate molecules needed for the support of cellular viability andorgan function, formulas are provided that permit cells, tissues andorgans to be preserved, for example, for at least 48 hours of staticcold storage at 0° C. without the need to add expensive ingredientsbefore use or to remove the preservative solution from the organ beforetransplantation. A range of effective concentrations of polyglycerol,alpha lactose, and other important components is provided.

In addition to polyglycerol and alpha lactose, additional valuablecomponents include chondroitin sulfate and chlorpromazine. Chondroitinsulfate is a connective tissue polysaccharide never before contemplatedfor use in organ preservation solutions. It has been used in highconcentrations to preserve corneas (e.g., “Optisol” contains 2.5% w/vchondroitin sulfate [Arch. Ophthalmol. 109: 864-868, 1991]), and it hasbeen used as a cryoprotectant (an agent that protects against damageincurred by freezing and thawing) in blood vessel segments submerged ina cryoprotectively effective concentration of glycosaminoglycans incombination with a cell penetrating cryoprotectant in a medium forfreezing blood vessels (U.S. Pat. Nos. 5,145,769 and 5,158,867), but ithas never been considered to improve the preservation of whole organsduring hypothermic storage. In a 1994 U.S. patent (U.S. Pat. No.5,306,711), for example, Peter Andrews reviews cornea preservationmedia, listing their contents of chondroitin sulfate, but then claimsorgan preservation solutions devoid of chondroitin sulfate, showing thelack of obviousness of including this agent in an organ preservationsolution. Chondroitin sulfate was used in one paper in 1979 in anattempt to modify immunogenicity after kidney transplantation (L. H.Toledo-Pereyra and V. R. Ramakrishnan, Transplantation 27(6):427-9,1979), but these authors found no reduction of hypothermic preservationinjury using 0.025% chondroitin sulfate, stating “there were nosignificant functional differences between the control and CIS[chondroitin sulfate]-treated groups,” which teaches away from the useof chondroitin sulfate in organ preservation perfusates or flushsolutions. We believe these experiments used a preservation model thatwas not appropriate for demonstrating beneficial effects of chondroitinsulfate or any other improvement in organ preservation solutions. In thepresent invention, the most preferred concentrations of chondroitinsulfate are 0.1-0.5% w/v, concentrations that are far lower than theconcentrations used for cornea hypothermic preservation or blood vesselfreezing (2.5% w/v).

Jabcobsen (Organ Preservation, Basic and Applied Aspects, D. E. Pegg, I.A. Jacobsen, and N. A. Halasz, editors, MTP Press, Lancaster, 1982, page324) found that chlorpromazine incorporation into a storage solution forrabbit kidneys was lethal after 24-48 hrs of storage when chlorpromazinewas used at a concentration of 15 micrograms/ml. Therefore, the abilityof chlorpromazine to strongly improve the efficacy of the currentinvention at a concentration of, for example, 5 micrograms/ml is mostsurprising. Because the use of chlorpromazine is believed to beunnecessary when good preservation solutions are used, or ifchlorpromazine is given to the donor before organ collection (see OrganPreservation, Basic and Applied Aspects, D. E. Pegg, I. A. Jacobsen, andN. A. Halasz, editors, MTP Press, Lancaster, 1982, pages 323-324), thevalue of including chlorpromazine in an organ preservation solution hasnot been recognized for the past 20 years or so.

RPS-2 is an excellent solution for static (non-perfusional) storage ofkidney slices and whole rabbit kidneys for relatively limited periods(see for example Khirabadi and Fahy, Cryobiology 30:10-25, 1994).Although RPS-2 causes no cellular injury as judged by sodium andpotassium transport capacity following cold storage for up to 4 days,its reliance on glucose as the main impermeant permits cell swellingeven after 24 hrs of cold storage, and therefore it is not ideal forlong term whole organ cold storage. By replacing glucose in RPS-2 withless permeable species, a first test of the acceptability of suchspecies based on lack of impairment of ion translocation followingstorage can be obtained.

As described in U.S. patent application Ser. No. 09/726,857, (hereinincorporated by reference), PGL can be used for prolonged tissue slicepreservation in a conventional 0° C. organ flush/cold storage solutionwithout detrimental effects, showing the utility of PGL for eitherstorage below 0° C. or for conventional storage at 0 to 10° C. As shownin FIG. 1, kidney slices stored in RPS-2 (curve labeled “Glucose”, opencircles) could be stored for 3 days near 0° C. with no demonstrabledeterioration based on their subsequent ability to accumulate potassiumand to extrude sodium during incubation in Cross-Taggart solution (seeFahy et al., in “Cell Biology of Trauma”, J. J. Lemasters and C. Oliver,Eds., CRC Press, 1995, pp. 333-356, and citations therein, for theprecise methodology of the functional assay). RPS-2 contains 180 mMglucose as a major component (and also includes 7.2 mM K₂HPO₄, 1 mMCaCl₂, 2 mM MgCl₂, 5 mM reduced glutathione, 28.2 mM KCl, 10 mM NaHCO₃,and 1 mM Adenine HCl). When 170 mM glucose was replaced with anosmotically equivalent amount of decaglycerol (black circles, curvelabeled “decaglycerol”), there was no significant difference between theK⁺/Na⁺ ratio obtained and the ratio obtained without glucose replacement(p>0.05). Polyethylene glycol of mean molecular mass ˜1000 daltons(PEG-1000, open boxes) also yielded good results, whereas sucrose (opentriangles) was distinctly inferior, and polyvinyl alcohol (blackhexagons) was overtly toxic. It is expected that PGL containing at least2 glycerol monomers (n>=2) will be effective for prevention of at leastshort-term (<=24 hr) cold storage injury, with 6 or more glycerolmonomers (n>=6) being generally preferred, and 10 or more monomers beingmost preferred (e.g., n=10 to 200) for simple cold storage.

The example of FIG. 1 underscores the lack of toxicity of even highconcentrations (170 mOsm) of PGL during prolonged contact with livingtissue. In the present application, this ability of polyglycerol tostabilize cell viability under conditions of reduced metabolism has beenexpanded by finding specific compositions containing polyglycerol thatpreserve not just tissue slices but also whole organs for prolongedperiods, and that surprisingly do so more effectively that the bestalternative organ preservation solution so far known in the art.

The lack of toxicity of a low concentration of PGL for a whole kidneywas shown by perfusing two kidneys for 5 hours at about 3.5° C. with asolution known as LM5 (the formula for LM5 was as disclosed in U.S.Provisional application 60/221,691, filed Jul. 31, 2000, and in U.S.patent application Ser. No. 09/916,396, filed Jul. 27, 2001, both ofwhich are herein incorporated by reference) to which was added 1% w/vdecaglycerol and other non-toxic polymers. The kidneys weretransplanted, and their recovery was measured by the postoperative serumcreatinine levels attained. As indicated in FIG. 2, the postoperativefunctional recovery of these kidneys was good, showing the lack oftoxicity of PGL for the vascular system during 5 hr of continuousperfusion and the applicability of PGL for use in perfusates, includingperfusates designed to contain cryoprotectants that are made to vitrifywith the assistance of the included PGL.

One embodiment of the preservation solution is a solution comprisinglactose, with or without other additives. A further embodiment is apreservation solution comprising a combination of polyglycerol andlactose, with or without other valuable additives. This is illustratedby means of the following Examples, which demonstrate the efficacy ofthe invention. In addition, a method of preserving cells, tissues andorgans using the disclosed preservation solutions at various temperatureis disclosed. In addition, a method of rescuing and preserving cells,tissues, or organs using a solution comprising polyglycerol at highertemperature, such as at room temperature or at body temperature, isdisclosed.

As is shown in the examples which follow, polyglycerol and alphalactose, two entirely novel candidate impermeants, were successfullytested and were found to have applications for conventionalpreservation. In particular, the use of alpha lactose or polyglycerolplus alpha lactose is useful for cell stabilization at about 0° C.±10°C. This includes isolated cells, cells in isolated tissues, or cells inorgans. Polyglycerol is useful for the control of cell swelling athigher temperatures as well, for example in applications relating tostroke, shock, cardiac arrest, hypoxia, poisoning, head trauma, brainswelling, inflammation, and other conditions in which cell swelling maybecome an important pathological outcome.

The Examples show unequivocal maintenance of cell and organ integrityand indicate that the combination of polyglycerol plus lactose, with orwithout other efficacious additives, is useful for the hypothermicpreservation of a wide range of cells from a wide range of species, andfor the treatment of other states that, like hypothermia, result in animpaired ability of cells to offset their tendency to swell underconditions of impaired metabolism or impaired cell volume homeostasis.

EXAMPLES Example 1

Lactose is Effective at Supporting Cellular Viability during HypothermicStorage

Table 1 shows that, in analogy to the results shown in FIG. 1, lactosecan support the ion translocation capacity of rabbit renal corticalslices stored at 0° C. TABLE 1 Exposure of Rabbit Renal Cortical Slicesto Test Solutions at 0° C. for about 5 Hours^(a) Test solution K⁺/Na⁺RPS-2 5.33 ± 0.20 RPS-Lactose 5.15 ± 0.22^(a)RPS-Lactose consists of RPS-2 in which about 175 mM glucose isreplaced with an equal number of millimoles/liter of lactose. Thedifference between the groups is not significant, which means thatlactose is an effective impermeant and is non-toxic to renal tissue.

Example 2

Successful 24-Hr Kidney Preservation withPolyglycerol/Lactose-Containing Solutions

FIG. 3 shows a comparison between the effects of 24 hrs of storage ofrabbit kidneys at 0° C. after flushing with UW Solution, a preferredcomposition containing decaglycerol and alpha lactose (Formula 1), and apreferred composition containing decaglycerol, alpha lactose, and 5micrograms/ml chlorpromazine (Formula 2; see Table 2 for the formulas ofthe solutions tested in FIG. 3) (means±1 SEM). We used chlorpromazineHCl for injection (Elkins-Sinn, Cherry Hill, N.J.) in these experiments.Kidney integrity was measured by following postoperative serumcreatinine levels. The model involved removing and preserving the rightkidney and transplanting it into the original donor at the site of theleft kidney after storage, with removal and disposal of the left kidneyat the time of transplantation. The ability of the model to detect anddiscriminate between different levels of reduced renal function wasaugmented by withholding hydration postoperatively, a maneuver which isbelieved to increase peak serum creatinine by about 2.5-fold.

In the experiments shown in FIG. 3, UW Solution was used exactly asdirected by the manufacturer, and the UW Solution used was obtained froma vendor (Barr Laboratories, Pomona, N.Y.) to ensure authenticity. UWwas washed out of the rabbit kidneys just prior to their transplantationusing 20 ml of lactated Ringer's solution. Control experiments showedthat this washout procedure did not affect the results of using Formula1 and therefore the procedure appears innocuous. Formula 1 was used byincorporating chlorpromazine (50 micrograms/ml) into the first 50 ml ofa flushing solution of Formula 1, and then washing the chlorpromazineout of the kidney prior to storage by using another 50 ml flush withFormula 1 devoid of chlorpromazine. In the case of Formula 2, thechlorpromazine was built into the solution and its concentration (5micrograms/ml) was not different during flushing and during storage.Neither Formula 1 nor Formula 2 were washed out of the rabbit kidneysbefore transplantation.

Surprisingly, the kidneys preserved with polyglycerol-lactose solutionsperformed unequivocally better than those preserved with UW Solution(VIASPAN), and the best results were obtained when storage took place inthe presence of the combination of decaglycerol, alpha lactose, andchlorpromazine, a combination which allowed essentially all injury to beeffectively suppressed. A useful concentration range for chlorpromazineis 1-50 micrograms/ml, with the most preferred concentration range being2-10 micrograms/ml. The superiority of chlorpromazine as an additive asin Formula 2 is surprising. But even without this additive, thedecaglycerol-alpha lactose solution shown in FIG. 3 produced nopostoperative serun creatinine values higher than 3.5, whereas over halfof the transplants using UW Solution resulted in creatinines exceeding4.0 (p<0.05 by Chi Square test). The p value for the comparison of themean peak creatinines obtained with UW Solution and Formula 1 was 0.014.Since current clinical practice avoids the storage of human kidneys formore than 24 hrs prior to transplantation, superiority to UW Solutionafter 24 hrs of storage is of direct clinical significance. We are notaware of other solutions this superior to UW after 24 hr.

Furthermore, there appeared to be a defect in reabsorptive capacity onthe part of UW Solution kidneys as compared to the decaglycerol/lactosekidneys, based on higher urine outputs, as determined from urine outputscoring, in the face of higher serum creatinine concentrations on day 1postoperatively in the UW group. This interpretation is supported by theclinical observation that the urine produced by experimental kidneys hada normal yellow color on day 1 whereas urine produced by UW-preservedkidneys appeared colorless, implying lack of concentration. TABLE 2Formulas for Some Preferred Embodiments of the Invention Formula Number1 1 2 2 3 3 4 Concentration Units mM or mM or mM or mM or mOsm g/litermOsm g/liter mOsm g/liter mOsm Physiologic support Ingredients:Adenosine 0 0 0 0 0 0 0 Adenine HCl 1 0.17 1 0.17 1 0.17 1 Glutathione,reduced 5 1.54 5 1.54 5 1.54 5 Na-acetate*3H₂O 2 0.27 2 0.27 2 0.27 2K₂HPO₄*3H₂O 7 1.6 7 1.6 7 1.6 7 KH₂PO₄ 0 0 0 0 0 0 0 NaCl 40  2.34 40 2.34 40  2.34 40  CaCl₂ 1 0.111 1 0.111 1 0.111 1 MgCl₂ 1 0.095 1 0.0951 0.095 1 MgSO₄*(6H₂O) 0 0 0 0 0 0 0 Impermeant ingredients: Glucose 25 4.5 25  4.5 25  4.5 25  90% decaglycerol ˜62*  20 ˜62*  20 ˜62*  20˜62*  Alpha lactose 45  16.2 45  16.2 45  16.2 45  Tripotassiumcitrate*H₂O 18  5.84 18  v5.84 18  v5.84 18  HES (pentafraction) 0 0 0 00 0 0 Lactobionic acid 0 0 0 0 0 0 0 Raffinose pentahydrate 0 0 0 0 0 00 Special additives: Chondroitin sulfate A 0 0 0 0 ˜20-0 1 0 200 μMChlorpromazine 0 0 14 × 10⁻⁶ 0.005  14 μM 0.005 14 μM HCl 0 0 0 0 0 0 0Allopurinol 0 0 0 0 0 0 0 Potassium hydroxide 0 0 0 0 0 0 0 PenicillinG*** 0 0 0 0 0 0 0 Regular insulin*** 0 0 0 0 0 0 0 Dexamethasone*** 0 00 0 0 0 41 μM Some Solution characteristics: Total sodium 42  42  42 42  Total potassium 68  68  68  68  Total chloride 41  41  41  43 Impermeant osmolality 186  186  186  186  PGL/lactose ratio   1.38  1.38   1.38   1.38 Total osmolality* 300  300  ˜302   ˜300   adjust topH:   7.4   7.4   7.4   7.4 Formula Number 4 5 5 6 6 UW SolutionConcentration Units mM or mM or MM or g/liter MOsm g/liter MOsm g/literMOsm g/Liter Physiologic support Ingredients: Adenosine 0 0 0 0 0 5  1.34 Adenine HCl 0.17 1 0.17 1 0.17 0 0 Glutathione, reduced 1.54 51.54 5 1.54 3    0.922** Na-acetate*3H₂O 0.27 2 0.27 2 0.27 0 0K₂HPO₄*3H₂O 1.6 7 1.6 7 1.6 0 0 KH₂PO₄ 0 0 0 0 0 25   3.4 NaCl 2.34 55 3.21 70  4.09 0 0 CaCl₂ 0.111 1 0.111 1 0.111 0 0 MgCl₂ 0.095 2 0.19 20.19 0 0 MgSO₄*(6H₂O) 0 0 0 0 0 5   1.23 Impermeant ingredients: Glucose4.5 25  1.8 10  1.8 0 0 90% decaglycerol 20 ˜31*  10 ˜31*  10 0 0 Alphalactose 16.21 40  14.41 25  9.01 0 0 Tripotassium citrate*H₂O 5.84 20 6.49 20  6.49 0 0 HES (pentafraction) 0 0 0 0 0 50  Lactobionic acid 0 00 0 0 100  35.83 Raffinose pentahydrate 0 0 0 0 0 30  17.83 Specialadditives: Chondroitin sulfate A 0 0 0 0 0 0 0 Chlorpromazine 0.005 0 00 0 0 0 HCl 0 0 0 0 0 0 0 Allopurinol 0 0 0 0 0    0.136 Potassiumhydroxide 0 0 0 0 0 100   5.61 Penicillin G*** 0 0 0 0 0 0 2 × 10⁵ UnitsRegular insulin*** 0 0 0 0 0 0    40 Units Dexamethasone*** 0.016 0 0 00 0    0.016 Some Solution characteristics: Total sodium 57  72  29Total potassium 74  74  125 Total chloride 62  77  0 Impermeantosmolality 156  126  −230 PGL/lactose ratio   0.78   1.24 NA Totalosmolality* 300  293  >320 Adjust to pH:   7.4   7.4 7.4*Concentration in milli-osmoles/kg (mOsm)**In UW Solution, “glutathione” = reduced glutathione plus oxidizedglutathione.***Must be added immediately prior to use, except for Formula 4.

Both of the solutions of the preferred embodiment in FIG. 3 contain 2%w/v decaglycerol, which imparts an osmotic contribution of about 62mOsm, and 45 mM alpha lactose, which in principle adds about another 45mOsm of impermeant osmotic pressure to the solution. The ratio of theosmotic contribution of decaglycerol to lactose is 62/45=1.38.Tripotassium citrate is present primarily as a buffer but is a nominalimpermeant as well, which, although it is known to penetrate cellsslowly, nominally contributes about 54 mOsm and brings the totalimpermeant osmotic pressure to 161 mOsm. If one considers glucose,another nominal impermeant (a slowly-permeating solute) to beeffectively impermeable over 24 hrs, its contribution of 25 mM bringsthe total impermeant concentration to 186 mOsm. The remaining componentsof the solution bring the total osmolality of the solution to 300 mOsm,which is close to the osmolality of blood. It is desirable to maintain asolution osmolality close to that of blood to avoid osmotic fluid shiftsbetween the organ and blood at the time the preserved organ istransplanted.

The osmolality of blood is about 290 mOsm. A 10% increment or decrementfrom this value is acceptable, i.e., a reasonable tonicity range is260-320 mOsm. At least one organ preservation solution in the past(Sack's solution) was successful for short-term preservation at totalosmolalities of around 400 mOsm, but this is not desirable because whenthe organ is transplanted, water will shift from the blood to the organ,causing edema and reducing total blood flow. It is envisioned that thepreferred upper limit of total osmolality is about 350 mOsm, or morepreferably about 320-330 mOsm. Ringer's solution, which has been usedfor very short term (hours) preservation, has an osmolality of about 260mOsm. A more preferred lower limit on total osmolality is believed to beabout 270 or more preferably 280 mOsm, because a lower osmolality maycause cells to swell too extensively on contact, which reduces theability of the organ to be perfused with the preservation solution andmay slow initial blood reflow upon transplantation.

As noted above, the manufacturer's instructions for the UW Solution,which say to flush the kidney until the blood has been fully removed,were followed in the experiments depicted in FIG. 3. For thedecaglycerol/lactose solutions, a standardized flush volume of 100 mlwas used to avoid changing the volume established in the initial seriesinvolving 50 ml with chlorpromazine followed by 50 ml withoutchlorpromazine (Formula 1). This 100 ml volume was not required to flushall the blood from the kidney, but was used as a standard. However, withUW Solution, even 100 ml was frequently insufficient for complete bloodwashout. We recorded the time required to flush the kidneys with UWsolution versus Formula 1, the volume of UW required for blood washout,and the mean flow rates (volume/time) for both solutions.

The results of these measurements on blood washout rates are given inTable 3. TABLE 3 Faster and More Efficient Blood Washout withLactose-Polyglycerol Solutions Time to Complete Solution Flushing Ratethe Flush Volume Used UW Solution 10.2 ± 0.62 ml/min 12.3 ± 0.98 min125.6 ± 9.31 ml Formula 1 18.1 ± 1.50 ml/min 5.65 ± 0.44 min 100.0 ± 0ml p value of <.001 <.001 differenceMeans ± SEM.

Table 4 provides a comparison between the effects and properties of thenew solutions described above versus those of UW Solution, the effectsbeing summarized with respect to results obtained after storage for 24hr. TABLE 4 Comparison Between Decaglycerol-alpha Lactose Solutions andUW Solution (VIASPAN), Including Functional Comparisons after 24 Hrs of0° C. Storage Formulas 1 and 2 UW Solution 1. Low viscosity 1. Highviscosity 2. Blood washout fast and 2. Blood washout slow and easydifficult 3. No additives before use 3. Needs 3 additives before use 4.No washout needed before 4. Must be washed out before transplanttransplant 5. No early failure to reabsorb 5. Early defect inreabsorption filtrate (early urine (early urine dilute, watery)concentrated, yellow) 6. Consistently low serum 6. Serum creatinine highin 5/9 creatinine cases 7. Mild organ shrinkage 7. Excessive organshrinkage 8. Inexpensive components that 8. Expensive components thatare are easy to dissolve hard to dissolve 9. Easy to filter-sterilize 9.Hard to filter-sterilize 10. Approximately isosmotic 10. Hyperosmotic toblood to blood

Example 3

24-Hr Preservation with Different Polyglycerol/Lactose Ratios

FIG. 4 shows the results of using solutions containing polyglycerolonly, lactose only, or both for 24 hr storage (means±SEM). Thesesolutions were Formula 1 (middle point, at a PGL content of 57.9% of thetotal of (PGL+lactose) (n=11); Formula 1 minus lactose, with the amountof decaglycerol raised from the 18 grams/liter found in Formula 1 (20grams of 90% decaglycerol stock=18 grams) to 32 grams/liter (35.5 g/l of90% stock), total osmolality=297 mOsm (point shown at 100% PGL, n=6);and Formula 1 minus decaglycerol, with the amount of lactose raised to102 mM (point shown at 0% PGL, n=5), total osmolality=300 mOsm.Intriguingly, although both the lactose-based solution and thedecaglycerol-based solution yielded results equivalent to or better thanUW solution (mean value for UW is represented as a solid horizontalline; dotted horizontal lines indicate plus and minus one standard errorof the mean of 10 observations), only the mixture of PGL and lactoseyielded a mean peak serum creatinine value that was statisticallysignificantly different from UW solution (as noted above, p=0.014 forthis comparison). The lactose-based solution yielded excellent resultsin 3 out of the 5 transplants (peak creatinines of 2-3, which issubstantially better than with UW solution), but only the addition ofPGL eliminated occasional high peak creatinines in a substantial numberof trials (n=11). The reason for the special effectiveness of thecombination of lactose and PGL is unknown, but the effect is clearly ofpractical importance.

Example 4

Successful 48-Hr Preservation with Polyglycerol-Lactose Solutions

All of the following experiments were carried out using a 48 hr storageperiod, but otherwise following the same transplantation protocol asdescribed above. FIG. 5 shows a comparison between the effects of 48 hrof storage of rabbit kidneys at 0° C. after flushing with UW Solution,Formula 2, Formula 3, or Formula 3B. As noted in Table 2, Formula 3 wasthe same as Formula 2, except for the addition of 0.1% w/v chondroitinsulfate A (catalogue number C 8529, Sigma Chemical Company; 70%chondroitin sulfate A and 30% chondroitin sulfate C, 0.1% w/v is thetotal concentration of the mixture). Formula 3B is the same as Formula3, except that the total concentration of chondroitin sulfate is 0.5%w/v rather than 0.1% w/v. The symbols in FIG. 5 represent means±1standard error of the mean.

For recovery days 3-8, Formula 2 gave consistently lower serumcreatinine levels than did UW Solution, though the differences were notstatistically significant. Adding chondroitin sulfate (Formulas 3 and3B) substantially shortened the time required to return creatinine to 2mg/dl (12 days for UW Solution vs. 7 days for Formulas 3 and 3B). Forthis reason, by day 6, both concentrations of chondroitin sulfateresulted in mean serum creatinine levels that were about 60% lower thanfor UW Solution. Formula 3 also lowered mean peak creatinine values fromover 10 for UW Solution to about 8. Based on these results, the range ofuseful concentrations of chondroitin sulfate extends from less than 0.1%w/v, such as 0.01% to 0.09%, to greater than 0.5%, such as 0.9% to 1%w/v. To our knowledge, no report of the successful use of chondroitinsulfate for organ preservation has ever appeared before.

We also tested the effects of adding dexamethasone (at 16 mg/liter) toFormula 2 (Formula 4, Table 2). The resulting curve of mean serumcreatinine values after 48 hrs of storage was virtually superimposableon that of the Formula 2 curve shown in FIG. 5, but there was lessvariation from experiment to experiment, which is considered a desirableeffect.

Clearly, at both 24 hrs and 48 hrs of preservation, there is asubstantial advantage of the new lactose-polyglycerol solutions over thecurrent industry standard,

Example 5

Effective Concentrations of Polyglycerol, Lactose, and Other Impermeants

Experiments were done to compare the results of storage for 48 hrs at 0°C. in lactose-polyglycerol solutions containing varying total impermeantspecies osmolalities (Formulas 1, 186 mOsm of impermeants; Formula 5,156 mOsm of impermeants; and Formula 6, 126 mOsm of impermeants; seeTable 2 for compositions). There were no statistically significantdifferences between the peak creatinine levels of the various groupsdespite wide variations in the total amount of impermeant species in thesolution, nor did these groups differ significantly from UW Solution.This indicates that lactose-polyglycerol solutions can be effective overa considerable range of total impermeant species concentrations. If oneconsiders that, after 48 hrs, glucose and citrate will have permeatedfully, the range of acceptable concentrations of truly (not justnominally or temporarily) impermeant species concentrations is evenbroader than indicated above. For Formula 6, subtracting thecontributions of citrate and glucose lowers total impermeantconcentration from 126 mOsm to just 56 mOsm.

The preferred upper limit of the impermeants in the solution is about250 mOsm, and even more preferably about 230 mOsm. The lowest limit ofthe impermeants in the solution is in principle about 20 mOsm, based onthe classical observation that 20 mOsm is the approximate value for thetotal impermeable solute osmotic concentration in a typical cell. Thus,under idealistic conditions, the lower limit may be as low as 20 mOsm.Under more common conditions, the lower limit may be closer to 40 mOsmor, more preferably, about 50 mOsm, or, based on FIG. 6, as high as 126mOsm, including 60, 70, 75, 80, 90, 100, 105, 110, 115, 120 and 125mOsm. Thus, the permissible total concentrations of lactose orpolyglycerol, when either species is present as the only impermeant, isabout 20-250 mOsm when the solution is being used for hypothermicpreservation of healthy cells, tissues, or organs.

When a solution of lactose or polyglycerol is being used to reverse warmischemic injury, as in resuscitation following cardiac arrest, higherconcentrations are generally appropriate. The typical upward osmoticpressure limit for a cell or a vascular bed, including the cerebralvasculature, is about 1500 mOsm. Therefore, for use in correctingischemic injury, in which water may need to be withdrawn from swollenparenchymal and endothelial/vascular cells in order to restore tissueperfusion, a concentration of polyglycerol, lactose, or a combination ofthe two may be prepared and used such that, then the solution is dilutedas a result of instillation into the area requiring assistance, itstotal concentration at the site of its action is up to 1500 mOsm.

Example 6

Efficacy of Formula 3 for Canine Kidney Preservation

Three canine kidneys were flushed with Formula 3 plus another ingredientand stored by simple cold storage. The rate of damage accumulationduring cold storage was estimated based on serum creatinine levels, andthis rate of damage accumulation (mg/dl of serum creatinine elevationper day of storage) was compared to the rate of damage accumulation forcanine kidneys flushed with and stored in UW Solution plus the sameextra ingredient. The result showed that the rate of damage accumulationin the Formula 3 group was essentially identical to that in the UWgroup, and there is no reason to believe that the extra ingredientaffected this comparison. Thus, Formula 3 is equivalent to UW Solutionin a canine model at least out to 48 hrs of cold storage at 0° C. From apractical point of view, this makes Formula 3 superior to UW, sinceFormula 3 is more convenient to use (see Table 4, which applies also toFormula 3 in regard to utilitarian aspects). The utility of Formula 3for both canine kidneys and rabbit kidneys, and its superiority to UWfor preserving rabbit kidneys, provides strong evidence that the formulawill be effective for human kidney preservation to at least 48 hrs,which is a clinically very significant period, and supports thepreferability of the solutions of the preferred embodiments to the useof UW Solution.

1. A method for the preservation of cells, tissues, or organs underconditions of impaired cell volume homeostasis, said method comprisingcontacting said cells, tissues, or organs with a solution comprisingpolyglycerol in an amount effective to preclude or to reverse cellswelling.
 2. The method of claim 1 wherein said polyglycerol is 2 to 200monomers in length.
 3. The method of claim 1 wherein the solution is ata temperature between about −10 and +10° C.
 4. The method of claim 1wherein the solution is at a temperature between about 0 and +10° C. 5.The method of claim 1 wherein the solution is at a temperature up toabout 37° C.
 6. The method of claim 1 wherein said contacting is viaintravenous or intra-arterial administration.
 7. The method of claim 1wherein said contacting is in vivo via arterial organ perfusion orretrograde venous perfusion of an organ or vascularized tissue.
 8. Themethod of claim 1 wherein said contacting is in vitro via arterial organperfusion or retrograde venous perfusion of an organ or vascularizedtissue.
 9. The method of claim 1 wherein said contacting is via theimmersion of or bathing of affected cells, tissues, or organs.
 10. Themethod of claim 5 wherein said polyglycerol is at a concentration offrom about 20 mOsm to 1,500 mOsm when in contact with said cell, tissue,or organ.
 11. The method of claim 5 wherein said preservation solutionfurther comprises lactose.
 12. The method of claim 11 wherein saidlactose is alpha lactose.
 13. The method of claim 5 wherein saideffective amount is an isotonic solution.
 14. The method of claim 5wherein said effective amount is a hypertonic solution.
 15. The methodof claim 1, wherein said cell swelling is related to one or more ofstroke, shock, heat stroke, warm ischemia, cardiac arrest, hypoxia,poisoning, head trauma, brain swelling, and inflammation.
 16. A methodfor the preservation of cells, tissues, or organs under conditions ofimpaired cell volume homeostasis, said method comprising contacting saidcells, tissues, or organs with a solution comprising decaglycerol in anamount effective to preclude or to reverse cell swelling.
 17. A methodfor the preservation of cells, tissues, or organs under conditions ofimpaired cell volume homeostasis, said method comprising contacting saidcells, tissues, or organs with a solution comprising lactose in anamount effective to preclude or to reverse cell swelling.
 18. The methodof claim 17 wherein the lactose is alpha lactose.
 19. The method ofclaim 17 wherein the lactose is at a concentration from about 11 mOsm toabout 1500 mOsm when in contact with said cells, tissues, or organs. 20.The method of claim 17 wherein the solution is at a temperature up toabout 37° C.